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CD8+ Tumor-Infiltrating T Cells Are Deficient in Perforin-Mediated Cytolytic Activity Due to Defective Microtubule-Organizing Center Mobilization This information is current as and Lytic Granule Exocytosis of September 24, 2021. Sasa Radoja, Masanao Saio, David Schaer, Mythili Koneru, Stanislav Vukmanovic and Alan B. Frey J Immunol 2001; 167:5042-5051; ; doi: 10.4049/jimmunol.167.9.5042 Downloaded from http://www.jimmunol.org/content/167/9/5042

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. CD8؉ Tumor-Inﬁltrating T Cells Are Deﬁcient in Perforin-Mediated Cytolytic Activity Due to Defective Microtubule-Organizing Center Mobilization and Lytic Granule Exocytosis

Sasa Radoja,* Masanao Saio,* David Schaer,† Mythili Koneru,* Stanislav Vukmanovic,† and Alan B. Frey1*

Tumor-inﬁltrating lymphocytes (TIL) are well known to be functionally impaired typiﬁed by the inability to lyse cognate tumor cells in vitro. We have investigated the basis for defective TIL lytic function in transplantable murine tumor models. CD8؉ TIL

are nonlytic immediately on isolation even though they express surface activation markers, contain effector phase cytokine mR- Downloaded from NAs, and contain perforin and granzyme B proteins which are packaged into lytic granules. Ag-specific lytic capability is rapidly recovered if purified TIL are briefly cultured in vitro and tumor lysis is perforin-, but not Fas ligand mediated. In response to TCR ligation of nonlytic TIL in vitro, proximal and distal signaling events are normal; calcium flux is rapid; mitogen-activated protein/extracellular signal-related kinase kinase, extracellular regulatory kinase 2, phosphoinositide-3 kinase, and protein kinase C are activated; and IL-2 and IFN-␥ is secreted. However, on conjugate formation between nonlytic TIL and cognate tumor cells in vitro, the microtubule-organizing center (MTOC) does not localize to the immunological synapse, thereby precluding exocytosis http://www.jimmunol.org/ of preformed lytic granules and accounting for defective TIL lytic function. Recovery of TCR-mediated, activation-dependent MTOC mobilization and lytic activity requires proteasome function, implying the existence of an inhibitor of MTOC mobilization. Our findings show that the regulated release of TIL cytolytic granules is defective despite functional TCR-mediated signal transduction. The Journal of Immunology, 2001, 167: 5042Ð5051.

rowth of cancer in the authochthonous host elicits anti- Human antitumor immune responses do not usually correlate tumor immune response and is detected by both T and B with tumor remission but has motivated considerable research in cell antitumor immune responses (1). For example, im- cancer immunotherapy. Although a variety of protocols have G by guest on September 24, 2021 mune cells infiltrating human tumors are seen in many different proved successful in immunization of rodents to resist primary tumor types and, although the prognostic significance of infiltrates tumor challenges, only rarely have large preexisting tumors been 2 varies among tumor type, tumor-infiltrating lymphocytes (TIL) eradicated (6). The failure to cure established rodent tumors and contain tumor-specific T cells. Patients often also have tumor-re- the modest success of human experimental immunotherapies, to- active T cells in lymph nodes draining tumor sites and in the cir- gether with the realization that antigenic human cancers elicit im- culation. These sources of T cells have been used to study the Ag mune response but are usually not eliminated, has sparked interest specificity and frequency of T cell antitumor immune response and in understanding the basis for inadequate antitumor T cell immune to prepare T cell lines used both in experimental adoptive immu- responses (7). notherapy (2) and as the basis for tumor Ag identification by ex- The presence of TIL in human cancers demonstrates that tumor pression gene cloning (3). Humoral antitumor immune response Ags are expressed in situ that are capable of eliciting antitumor T has also been noted in many human cancers (4), and patient sera cell immune response, which nonetheless fails to eliminate tumors have similarly been useful in identification of tumor Ags, most ϩ (8). The lytic function of CD8 TIL has been investigated in var- recently by the SEREX method (serological analysis of recombi- ious human (9) and murine (10, 11) tumors and found to be de- nant tumor cDNA expression libraries; Ref. 5). Patient humoral fective. Deficient lytic function is transient because, on purification and T cell responses prove that human cancers are able to prime from tumor cells and culture in vitro, tumor-specific killing can be tumor-specific immune responses. Similar studies using experi- detected (12). mental models have proven the immunogenicity of rodent tumors. We have investigated the basis for defective TIL lytic function in transplantable murine tumor models. Freshly isolated TIL, al- Departments of *Cell Biology and †Pathology, New York University School of Medi- though nonlytic in vitro, contain mRNAs encoding effector phase cine, New York, NY 10016 cytokines, serine esterases, and perforin characteristic of cytolytic Received for publication June 22, 2001. Accepted for publication August 22, 2001. effector T cells. Furthermore, perforin and granzyme B proteins are The costs of publication of this article were defrayed in part by the payment of page synthesized and packaged into lytic granules showing that TIL are charges. This article must therefore be hereby marked advertisement in accordance ϩ with 18 U.S.C. Section 1734 solely to indicate this fact. mature CD8 T cells. TIL recover perforin-mediated, Ag-specific 1 Address correspondence and reprint requests to Dr. Alan B. Frey, Department of lysis if purified and cultured briefly in vitro. After recovery of lytic Cell Biology, New York University School of Medicine, Room MSB 690, 550 First function, on conjugate formation with cognate tumor cells in vitro, Avenue, New York, NY 10016. E-mail address: [email protected] TIL mobilize cytotoxic granules to the immunological synapse, 2 Abbreviations used in this paper: TIL, tumor-infiltrating lymphocyte; AICD, activation-induced cell death; MTOC, microtubule-organizing center; PKC, protein ki- whereas granules in freshly isolated, nonlytic TIL do not migrate. nase C; FasL, Fas ligand; PI3 kinase, phosphatidylinositol 3-kinase. In addition, the microtubule-organizing center (MTOC) does not

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 5043 localize to the immunological synapse in nonlytic TIL. This defect Conjugate formation and intracellular staining for fluorescence precludes movement of lytic granules to the immunological syn- microscopy apse and accounts for defective TIL lysis. Biochemical analyses CD8ϩ TIL (6 ϫ 105) were mixed with tumor cells (3 ϫ 105) in 0.6 ml demonstrated that TCR kinase-mediated signaling is functional in serum-free RPMI 1640 at room temperature. Cells were centrifuged at freshly isolated, nonlytic TIL. Our findings show that after arrival 170 ϫ g for 10 min to promote conjugate formation, incubated for 5Ð10 to the tumor microenvironment, antitumor T cells are unable to min at 37¡C, and transferred to coverslips (VWR Scientific, South Plain- release cytolytic granules on contact with tumor which may permit field, NJ) pretreated with 1% Alcian blue. Cells were fixed in 4% parafor- maldehyde for 30 min at room temperature, permeabilized with 0.01% tumor escape from T cell lysis. saponin, 0.5% BSA in PBS (30 min at room temperature), and then then stained with 0.5 ␮g/ml of mouse monoclonal anti-␤-tubulin Ab (Boehr- inger Mannheim, Indianapolis, IN), mouse anti-Rab 27a (BD Transduction Materials and Methods Laboratories, San Diego, CA), rabbit anti-PYK-2 (a gift from J. Schless- Mice inger, New York University School of Medicine), or goat polyclonal anti- granzyme B Ab (Santa Cruz Biochemicals, Santa Cruz, CA) (1 h at 37¡). Male C57BL/6 mice were obtained from The Jackson Laboratory (Bar After three washes (0.01% saponin, 0.5% BSA in PBS), cells were incu- Harbor, ME). Mice were housed four per cage in a barrier facility and bated with secondary Ab (FITC-conjugated anti-mouse IgG for ␤-tubulin maintained on a 12-h light/dark cycle (7 a.m. to 7 p.m.) with ad libitum and Rab 27a or PE- or FITC-conjugated anti-goat for granzyme B) (30 min access to food and water. A sentinel program revealed that the mice were at RT¡), washed three times (0.01% saponin, 0.5% BSA in PBS), incubated murine hepatitis virus negative and the tumor cell lines are murine hepatitis with 0.1% Evans blue for 30 s, and washed thoroughly. Preparations were virus negative. Experiments involving animals were conducted with the covered with mounting solution before fluorescence or confocal micros- approval of the New York University School of Medicine Committee on copy using a MRC 600 device (Bio-Rad, Hercules, CA). MRC images Animal Research. were converted to PICT files using mrc2 M software (version 1.9.3). Downloaded from Immunoblot analysis Tumors Activation of purified T cells in vitro and immunoblot analyses was per- MCA-38 adenocarcinoma (13), P-815 cells (14), and 6-1 fibrosarcoma (15) formed as described (12). Some immunoblotting experiments used whole 6 have been described recently (12). Then 1Ð2 ϫ 10 cells were injected s.c cell detergent extracts prepared from freshly isolated TIL (or TIL that were in 0.1 ml HBSS for tumor induction. recovered in vitro before extract preparation) which were not activated in vitro. http://www.jimmunol.org/ Tissue culture TIL subcellular fractionation and PKC analysis was performed essen- tially as described (17). Equal cell equivalents of soluble and membrane Sources and use of all tissue culture reagents have been described recently fractions were subjected to SDS-PAGE followed by immunoblotting with (12). isoform-specific Abs and detection with ECL (Amersham-Pharmacia Bio- tech, Little Chalfont, Buckinghamshire, England). Isolation of TIL Antibodies used for immunoblotting were: mouse monoclonal anti- phosphotyrosine, (clone PY20, Transduction Laboratories, Lexington, Isolation of TIL from tumors by immunomagnetic separation was recently KY), mouse monoclonal anti-ERK1 (clone MK1, Transduction Laborato- described (12). Potentially inhibitory effects of the immunobead isolation ries), polyclonal rabbit anti-active MAPK (pTEpY, Promega, Madison, protocol on TIL lytic function are unlikely because we have reisolated TIL WI), polyclonal rabbit anti-Akt and polyclonal rabbit anti-phospho-Akt by guest on September 24, 2021 with anti-CD8 magnetic beads after recovery in vitro, and those cells are (Ser473) (Cell Signaling Technology, Beverly, MA), mouse anti-Rab 27a, lytic. As a further control, we have prepared CTL by primary MLR in vitro rabbit anti-PYK-2, and anti-PKC isoforms ␣, ␦,or⑀ (Upstate Biotechnol- followed by isolation with anti-CD8 immunobeads. These cells are fully ogies, Lake Placid, NY), and ␤ (Transduction Laboratories). lytic, again showing that the isolation protocol is without effect on lytic activity. Intracellular calcium mobilization studies Flow cytometric intracellular staining of T cells CD8ϩ TIL were isolated and resuspended at 106 cells/ml in serum-free RPMI 1640. Cells were incubated with 1 ␮M Indo-1 (Sigma Chemical, St. Single-cell suspensions were prepared from tumors, primary MLR, or Louis, MO) for 1 h, washed three times, and stimulated (10 ␮g/ml anti- spleens of control mice by enzyme digestion and magnetic immunobeading ⑀ ␮ CD3 mAb (clone 500-A2) or 1 M ionomycin), and the OD398 and 480 was as recently described (12). For granzyme B analysis from all sources, T measured over time. Excitation was at 340 nM in a Fluoromax 2 spec- cells were isolated by positive selection using anti-CD8-conjugated beads. trofluorometer calibrated as described (18). For analysis of perforin expression in TIL, positive selection was also used. For granzyme B analysis of splenocytes, T cells were prepared by negative selection using anti-MAC1-, anti-CD4Ϫ, and anti-B220-conjugated beads. Flow cytometric analyses were performed as recently described (12). Pri- Results mary Abs (either species- and isotype-matched nonimmune Ig, rat anti- TIL do not exhibit ex vivo cytotoxicity but recover perforin-me- perforin (clone KM585; Kamiya Biochemical, Seattle, WA), goat anti- diated, tumor-specific cytolytic function after short term in vitro granzyme B (Santa Cruz Biotechnology, Santa Cruz, CA)) were used at 0.5 ␮g/106 cells followed by appropriate secondary Abs (goat anti-rat Ig culture (12). For these analyses we studied two tumors, a fibro- (Caltag Laboratories, Burlingame, CA) or donkey anti-goat (Jackson Im- sarcoma made by expression of cDNAs encoding dominant-neg- munoResearch Laboratories, West Grove, PA)). ative p53 plus activated Ha-ras genes in primary embryonic fibroblasts termed 6-1 (15), and separately a chemically induced Generation of MLR primary CTL in vitro adenocarcinoma, MCA-38 (13). For all experiments data achieved CTL were prepared from primary MLR as described (12). with either tumor were highly similar if not identical and repre- Chromium release assay sentative data is shown. Furthermore, all experiments were performed with TIL isolated from individual mice (i.e., not mixed 51 CTL activity was determined in standard Cr release assays as recently from different tumor preparations) and were repeated more than described (12). Redirected cytolysis assays, using anti-CD3⑀ Ab 145-2C11 ϩ (at 0.001 mg/ml final) and P-815 cells as targets, were performed as de- five times with equivalent results. CD8 TIL were purified by scribed (16). Hamster IgG was used as control Ab for redirected assay, and magnetic immunobeading from tumor single cell suspensions and lysis of labeled targets in the presence of effector cells plus control IgG was tested for lytic ability (Fig. 1A). Freshly purified TIL are not lytic equivalent to spontaneous release. Specific lysis was determined as de- in standard four hr assays, however, if TIL are cultured in vitro in scribed (12). Pharmacological inhibition of T cell kinases before lysis assay used the presence of IL-2, substantial lytic activity is detected. The level wortmannin, bisindolmaleamide, or PD98059 (all obtained from Calbio- of lysis is comparable to that demonstrated by primary MLR CTL chem, La Jolla, CA) at the dosages indicated in Fig. 6. at equivalent E:T ratios (Fig. 6). 5044 DEFECTIVE TIL MTOC MOBILIZATION

vitro (data not shown)). TIL isolated from a variety of other tumors were similarly tested for Ag-specific lysis after recovery of lytic function in vitro and in all cases TIL demonstrate tumor-specific lysis (fibrosarcoma 6-1 (15), EL4 thymoma (19), EG.7 (EL4 ex- pressing an MHC class I-restricted OVA peptide) (20), MC57G sarcoma (21), and mastocytoma P815 (14); data not shown). The mechanism of cytolysis in recovered TIL was tested by inclusion of either EGTA (which inhibits requisite functional po- lymerization of perforin, MTOC mobilization, and lytic granule exocytosis by chelating free calcium (22)), or blocking Ab to Fas ligand (FasL; Fig. 1C). Anti-FasL Ab had no effect, however, EGTA completely abrogated lysis showing that target cell killing was perforin-mediated. In addition, the rapid kinetics of in vitro cytolysis by recovered TIL is characteristic of perforin-, but not FasL-, mediated lysis (23). Collectively these experiments show that freshly isolated TIL are defective in tumor cell lysis in vitro but lytic function is restored after purification from tumor and brief culture in vitro. In addition, TIL lysis of cognate tumor cells is Ag-specific and Downloaded from perforin-mediated.

Recovery of TIL lytic function The recovery of TIL lytic function was examined. In preliminary experiments TIL were incubated in various concentrations of rIL-2

for different periods of time and cytolysis determined. As little as http://www.jimmunol.org/ 5 U/ml of rIL-2 for 12 h was sufﬁcient to permit maximal recovery of lytic function (data not shown). Furthermore, TIL could recover signiﬁcant lytic function if cultured in vitro for 6 h without additional medium supplements, typically 50Ð75% of maximal lysis (Fig. 2). Actinomycin D, cycloheximide, or brefeldin A had no effect on recovery of cytolytic function if included during the 6 h recovery period showing that recovery of lytic function does not require RNA, protein synthesis, or protein secretion. Recovery of by guest on September 24, 2021 maximal lytic activity required ϳ12 h of in vitro culture, both RNA and protein synthesis (data not shown), and exogenous IL-2.

TIL are mature CD8ϩ T cells One potential reason for the inability of TIL to lyse tumor target FIGURE 1. TIL do not exhibit ex vivo cytotoxicity immediately on iso- cells in vitro may be related to the state of TIL maturation which lation but recover tumor-specific, perforin-mediated lytic function after is reflected in the transcriptional activity of effector phase genes short term in vitro culture. A, Freshly isolated TIL are nonlytic. TIL were (24). The observation that protein or RNA synthesis is not required f isolated and either tested for lytic function immediately ( ) or cultured in for recovery of significant TIL lytic function suggested that TIL F medium containing 20 U/ml rIL-2 for 12 h before lysis assay ( ). Target are mature effector T cells. We characterized TIL further by ana- cells were cognate MCA-38 tumor cells in both assays. B, TIL exhibit lyzing expression of genes characteristic of the effector phase. Ag-specific lysis. TIL were isolated and cultured overnight in the presence ϩ of rIL-2 before cytolysis assays using either cognate MCA-38 tumor cells CD8 TIL were purified from tumors and used to prepare cDNA (f) or syngeneic MC57G tumor cells (F) as target cells. C, TIL cytolysis is perforin dependent. TIL were isolated and cultured overnight in the presence of rIL-2 before cytolysis assay using cognate MCA-38 tumor cells as targets. Cells were preincubated with no additives (f),1mM EGTA (F), or 10 ␮g/ml anti-FasL-blocking Ab (Œ), and additions were maintained during the 4-h lysis assay. Isotype-matched nonimmune Ig has no effect on lysis (data not shown).

Because of concern that the inflammatory milieu of the tumor microenvironment may attract T cells which does not represent specific recruitment of antitumor T cells, TIL were tested for Ag- specific lysis. Syngeneic MC57G tumor cells, which do not im- FIGURE 2. Recovery of TIL lytic function does not require either protein synthesis or secretion. TIL were purified and plated in vitro at a con- munize mice to resist challenge injections of MCA-38 (and there- centration of 106 cells/ml in the presence or absence of medium additions fore do not express cross-reactive tumor Ags), are not appreciably for either 6 or 12 h as indicated. rIL-2 was added at 20 U/ml. Actinomycin lysed by anti-MCA-38 TIL (Fig. 1B) (TIL were also tested for lytic D, brefeldin A, and cycloheximide were added at 5 ␮g/ml. After in vitro function by redirected lysis assay (in which Ag specificity is ir- incubation, cells were washed by centrifugation in complete RPMI me- 51 relevant) and found to be nonlytic showing that the lytic defect is dium, and cytolysis was determined using Na2 CrO4-labeled cognate tu- not caused by an unknown inhibitory MCA-38 tumor influence in mor cells as targets at an E:T ratio of 10:1. The Journal of Immunology 5045 for programming of RT-PCR analysis of cytokine gene transcripts. Collectively, these experiments demonstrate that TIL contain Freshly isolated CD8ϩ TIL contain mRNAs encoding IL-2, IFN-␥, both cytokine mRNAs (12) and cytolytic enzymes localized in perforin, granzyme A, and granzyme B (12). cytotoxic granules characteristic of mature CD8ϩ effector T cells. In addition, expression of granzyme B and perforin proteins in TIL was examined (Fig. 3A). As anticipated, primary MLR CD8ϩ TIL cytokine secretion T cells demonstrating high levels of lysis in vitro (compare Fig. 6) The RNA analysis showed that TIL contain cytokine mRNAs (12) contain both granzyme B and perforin. Freshly isolated, nonlytic but did not reveal whether these cytokines are secreted. To address TIL contain equivalent levels of perforin and slightly higher levels this, purified nonlytic TIL were stimulated in vitro and secretion of of granzyme B compared with MLR T cells. The level of perforin IL-2 and IFN-␥ assessed by ELISA (Fig. 4). Secretion of IFN-␥ protein in TIL does not change when TIL are cultured in vitro for was rapid: after2hofinvitro activation ϳ300 U/106 cells were 12 h (data not shown) suggesting that recovery of lytic function is produced. After4hofactivation, high levels of IFN-␥ were pro- not due to increased expression of perforin. duced, ϳ3000 U/106 cells. The demonstration of perforin and granzyme B proteins in freshly isolated, nonlytic TIL proves that TIL lytic deficiency in TIL-cytolytic granules do not localize to the immunological vitro is not due to the absence of these enzymes, which would synapse indicate that TIL are not fully mature, but does not address the For effector T cells to demonstrate lytic function in response to possibility that cytolytic granules are not formed or loaded with TCR-mediated signaling after target cell recognition, preformed lytic enzymes. To address this concern, we performed immunocy- cytotoxic granules must accumulate under the plasma membrane tochemical localization of perforin and granzyme B, both in frozen closely apposed to target cells, fuse to the membrane, and then Downloaded from thin sections of tumor (Fig. 3B) and in purified TIL (data not release granule contents. Because TIL contain perforin and serine shown), and found that perforin and granzyme B (Fig. 5A) proteins esterases but are nonlytic, we considered the possibility that gran- are localized in cytoplasmic granules. ule exocytosis was defective. We tested whether localization of lytic granules to the immunological synapse occurred on TCR- mediated recognition of target cells by redirected assay in vitro (Fig. 5A). TIL, either freshly isolated or after in vitro recovery, http://www.jimmunol.org/ were mixed with target cells for 5Ð10 min, and then the intracellular localization of lytic granules was determined by immunofluorescence microscopy using Ab to granzyme B. In freshly isolated, nonlytic TIL, only ϳ5% of T cells forming conjugates orient lytic granules to the area of tumor cell contact. However, in ϳ60Ð70% of conjugates formed between tumor cells and recovered TIL, granzyme Bϩ granules were reoriented to the immunological synapse. Representative pictures of granzyme B staining are shown in by guest on September 24, 2021 Fig. 5A, where in recovered but not freshly isolated TIL, lytic granules are easily visualized in close apposition with tumor cell targets.

FIGURE 3. TIL are mature CD8ϩ T cells. A, TIL contain perforin and granzyme B proteins. Single-cell suspensions were prepared, and CD8ϩ T cells were purified by magnetic immunobeading (positive selection from tumors and negative selection from spleens). T cells were labeled for either perforin or granzyme B and CD8. Flow cytometric analyses were gated on CD8ϩ T cells. TIL (solid tracings in right panel) or primary MLR T cells (dashed tracings in right panel) were isolated and analyzed by flow cy- tometry for perforin and granzyme B proteins. Binding of primary Ab was FIGURE 4. Cytokine secretion from purified TIL. TIL were isolated detected using fluorochrome-conjugated secondary Abs. Species- and isotype- and plated in triplicate (at 2 ϫ 105 cells/well) in 96-well dishes precoated matched control Abs were used to establish the levels of nonspecificAbla- with either anti-TCR (ϩ) or control Ab (Ϫ). At the indicated times, culture beling (shaded histograms). B, TIL perforin is localized in granules. Immu- supernatants were collected and assayed for IFN-␥ or IL-2 by ELISA. nocytochemical detection of perforin protein (arrows) was made in frozen thin Standard curves established using recombinant cytokines showed that the sections prepared from tumor tissue. There was no staining of cells using ELISA sensitivity was 19 pg/ml for IL-2 and 195 pg/ml for IFN-␥. One isotype-matched control Ab (data not shown). Magnification, ϫ100. nanogram IL-2 equals 37.5 U, and 1 ng IFN-␥ equals 5.2 U. 5046 DEFECTIVE TIL MTOC MOBILIZATION

apse, where they fuse to the plasma membrane to release lytic enzymes. Because TIL were determined to contain mature lytic granules and because we showed that mobilization of granules to the T cell immunological synapse was defective, we asked whether TIL MTOC localized to the area of cell contact on formation of conjugates with cognate tumor target cells. As was found by analysis of lytic granule migration, TIL MTOC did not localize to the immunological synapse (Fig. 5B). In TIL, the lytic function of is restored by purification and short term in vitro culture, the MTOC localizes to the immunological synapse within 5Ð10 min of contact with cognate tumor in vitro. Granule migration in recovered TIL does not occur if noncognate tumor is used as target cells (data not shown). This finding shows that nonlytic, freshly isolated TIL are defective in an obligate early step in the process of TCR-mediated release of preformed cytotoxic granules, that of MTOC mobilization. Furthermore, recovery of lytic function is correlated with MTOC reorientation to the immunological synapse. Downloaded from Conjugate formation by redirected assay does not restore lytic function or lytic granule mobilization Freshly isolated, nonlytic TIL were observed to form fewer conjugates with cognate target cells in vitro compared with TIL after recovery (Table I). One possible interpretation of this finding is

that target cell contact by TIL may be unstable, which might result http://www.jimmunol.org/ in insufficient activation of T cell lytic function. We consider this interpretation unlikely: when conjugates are formed between nonlytic TIL and target cells by bridging TIL and target cells with anti-CD3⑀ Ab (redirected assay), an equal number of conjugates are formed by nonlytic TIL as for lytic TIL, but cytolysis still does not occur (Table I). This finding shows that target cell conjugate formation is necessary but insufficient for cytolysis. Recovered TIL in the redirected conjugate formation assay are both lytic, and by guest on September 24, 2021 their lytic granules mobilize to the immunological synapse (Fig. 5A), but freshly isolated TIL are nonlytic and do not mobilize their lytic granules. Collectively, these observations show that even when nonlytic TIL form conjugates at the same frequency as lytic TIL, lytic granules do not localize to the immunological synapse.

FIGURE 5. Mobilization of cytotoxic granules is defective in TIL. A, Role of different protein tyrosine kinases in TIL lytic function TIL do not localize cytotoxic granules to the immunological synapse. TIL were isolated, used in redirected assays with P-815 cells as targets, and Release of granules from cytolytic cells involves a Ras-dependent analyzed by confocal microscopy using Ab to granzyme B either before and a Ras-independent pathway of signal transduction. NK cells recovery in culture or after as indicated. Nonimmune goat IgG was used as activated to release granules, either on recognition of MHC class control. Arrows, granzyme Bϩ granules. Identical results were obtained I-negative target cells or via Fc␥RIII ligation, use signal transduc- using cognate tumor cells as targets. B, TIL MTOCs do not reorient on tion involving MAPK-ERK, Vav, Pyk-2, PI3 kinase, and Syk 70 contact with target cells. TIL were isolated and used in reorientation assays with cognate MCA-38 cells either immediately on isolation or after overnight recovery. Cells were stained using anti-␤-tubulin and processed for immunofluorescence microscopy. Arrows, MTOC. Nonimmune murine Ig Table I. Freshly isolated, nonlytic TIL form fewer conjugates with was used as control. At least 10 microscopic fields per sample were cognate target cells in vitro compared with TIL after recovery viewed, each with 25Ð75 T cells per field, and this experiment was repeated eight times. In fresh, nonlytic TIL, of those TIL forming conjugates, only % TIL Forming Conjugates 5Ð15% of cells have reoriented MTOC (toward target cells). In recovered, lytic TIL, the percentage is 45Ð70%. Magnification, ϫ150. Cognate targets Redirected Expt. Nonlytic Lytic Nonlytic Lytic

1 5 22 36 38 Exocytosis of TIL lytic granules is blocked due to failure of the 2 6 20 36 36 MTOC to localize to the T cell immunological synapse 3 9 32 30 31 4 7 25 33 35 After TCR-mediated recognition of cognate Ag, migration of lytic a granules to the immunological synapse requires several sequential TIL were isolated and either tested for conjugate formation with P-815 cells or cognate tumor cells as targets immediately or after recovery in vitro. Aliquots were biochemical events including orientation of the MTOC to the T tested for lytic function, and conjugates were enumerated by light microscopy by cell immunological synapse. The MTOC is a multicomponent counting the number of conjugates per microscopic field and the total number of T cells in the same field. The average percentage of TIL forming conjugates was ob- structure that is the site for aggregation of cytoplasmic microtu- tained from viewing 5Ð10 microscopic fields each with 50Ð100 cells per field. Con- bules on which lytic granules migrate to the immunological syn- jugate formation by redirected assay was dependent upon anti-CD3␧ Ab. The Journal of Immunology 5047 kinases (25). Granule release in T cells is less well understood: one report shows that MAPK-ERK activation is required, thought to be activated primarily through the TCR pathway (26); whereas other reports implicate PI3 kinase (27) or PKC in lytic function (28). After recovery of TIL lytic function, the role of three major T cell protein tyrosine kinases in TIL lysis was investigated by inclusion of various inhibitors during lysis assays. For these experiments, the redirected assay was used, so that direct comparison of TIL function with primary MLR CTL T cells (used as positive controls) could be made under identical conditions. Because the results of lysis assays were consistent when conducted at different E:T ratios, for comparison of the effects of different inhibitors, only data from a single E:T ratio (10:1) is shown. Over a range of concentrations typically used in inhibition assays (27) inhibition of either PI3 kinase (wortmannin), ERK (PD98059), or PKC (bisindolmaleamide) caused dose-dependent reduction of lytic function (Fig. 6). MLR CTL lytic function was also inhibited in a manner similar to that for TIL, showing that the requirement for PI3 kinase, ERK, and PKC in target cell lysis is not restricted to TIL. Downloaded from

Proximal TCR-mediated signal transduction is normal in nonlytic TIL Using either NK cell or T cell lines, others have shown that pharmacological inhibition of ERK (29), PKC (28), or PI3 kinase (30) blocks lytic function. We analyzed TIL for the presence and acti- http://www.jimmunol.org/ vation status of those kinases suggested to play a role in granule release. First, we examined TIL for the pattern of total tyrosine- phosphorylated proteins by immunoblot analysis of lysates prepared from freshly isolated TIL before and after activation in vitro with anti-TCR Ab. As a control, spleen CD8ϩ T cells prepared by FIGURE 7. A, TIL have normal levels and pattern of total tyrosine identical procedures were used for comparison. Similar to spleen phosphoproteins, ERK2, and phosphorylated Akt after TCR ligation in cells, TIL have a very low basal level of tyrosine-phosphorylated vitro. TIL, primary MLR CTL, or splenocytes were isolated and either proteins and, on activation in vitro, several prominent protein spe- activated in vitro (ϩ) or not (Ϫ) as indicated. Detergent lysates were pre- by guest on September 24, 2021 cies are rapidly phosphorylated (Fig. 7A) including ZAP 70 and pared from cells and subjected to SDS-PAGE before transfer to nylon mitogen-activated protein/extracellular signal-related kinase ki- membranes. Membranes were then used for immunoblotting using Abs nase (data not shown). This observation shows that TIL proteins reactive with phosphotyrosine (␣PY), total and phosphorylated ERK proteins, and total and phosphorylated Akt protein as indicated. Freshly isolated (f) or recovered (r) TIL or MLR CTL were activated with anti-TCR (␣TCR) Ab and analyzed for ERK activation. B, PKC isoforms localize to the T cell plasma membrane after TIL TCR ligation in vitro. CD8ϩ T cells, TIL, or TIL after recovery in vitro were isolated by magnetic immunobeading as described previously. Cells were activated for the indicated times, and cell membrane or soluble fractions were prepared and analyzed by immunoblotting with Abs reactive to the indicated PKC isoforms as described in Materials and Methods.

are not hyperphosphorylated due to overstimulation in the tumor microenvironment, which might inhibit subsequent activation events, and also that TCR signal transduction is intact at least at the level of the proximal components of the pathway. Because ERK has been implicated in granule release from both T cells and NK cells we next examined the levels and activation status of ERK. Again, like spleen CD8ϩ T cells, freshly isolated TIL contain ERK1/2 protein (Fig. 7A). Stripping the immunoblot membrane and reprobing with Ab specific for activated (phosphorylated) ERK revealed enhanced phosphorylation of the p42 form of ERK (ERK2) in TIL, but not ERK1, in contrast to activated FIGURE 6. Role of different protein tyrosine kinases in TIL lytic func- spleen T cells which contain both ERK1 and ERK2. tion. TIL were isolated and recovered by overnight culture. TIL or primary On discovery that TIL do not contain p44 phospho-ERK, we MLR CTL were used in standard chromium release assays at an E:T ratio of 10:1 by redirected assay using P-815 cells as targets. Kinase inhibitors considered that the failure to activate both forms of ERK may be were added to T cells (at the concentrations indicated on the y-axis) 60 min the biochemical basis for defective MTOC orientation and lytic before the start of 4-h lysis assays. BIM, bisindolmaleamide. function. This possibility was further investigated by assessment of 5048 DEFECTIVE TIL MTOC MOBILIZATION the levels of ERK proteins in TIL whose lytic function was restored. Lysates were prepared from either freshly isolated or recovered TIL and, as positive control, purified CD8ϩ T cells from primary MLR cultures, and analyzed by immunoblotting. Lysates were prepared directly after purification or following activation of TIL in vitro with anti-TCR Ab. Aliquots of both recovered TIL and MLR cells were determined to be lytic (ϳ40% lysis at E:T of 10:1- data not shown). Nonlytic and lytic TIL and lytic MLR T cells have phospho-ERK2 but do not contain activated p44 ERK demonstrating, we believe for the first time, that ERK1 is not required for perforin-mediated lytic function in T cells and eliminating the hypothesis that the failure to activate p44 ERK in TIL is the basis for lytic dysfunction. Because activity of PI3 kinase has been shown to be involved in cytolytic function of NK cells (30) and T cells (Fig. 6), we asked whether PI3 kinase was activated in TIL. For this determination we analyzed the level and activation status of the immediate down- ϩ FIGURE 8. Calcium flux in TIL is normal. TIL were isolated, and cal- stream target of PI3 kinase, Akt. Primary MLR CD8 T cells, cium flux was determined in response to stimulation in vitro with either Downloaded from again used as positive controls, and freshly isolated TIL were pre- anti-CD3⑀ Ab (10 ␮g/ml) or ionomycin (1 mM) stimulation as indicated. pared and either activated or not before lysate preparation followed A portion of TIL were allowed to recover in vitro overnight in the presence by immunoblot analysis. TIL contain nonphosphorylated Akt pro- of rIL-2 before assay. tein which is rapidly phosphorylated on activation showing that TCR-mediated signal transduction through PI3 kinase is intact in nonlytic TIL. Collectively, these pharmacological and biochemical analyses

Because the inhibitor experiments implicated a role for PKC in demonstrate that the major signal transduction pathways in non- http://www.jimmunol.org/ CTL function and also because of a previous report suggesting the lytic TIL are intact and that the three protein tyrosine kinases sug- same requirement (28), activation of PKC was also tested. Cell gested to be essential in cytolytic function, ERK, PKC, and PI3 extracts were prepared either from freshly isolated or from TIL kinase, as well as calcium mobilization, are functional in TIL. recovered by in vitro culture. The subcellular localization of sev- TIL localization of PYK-2 or Rab 27a eral PKC isoforms was determined by fractionation of extracts into membrane and cytosolic fractions that were subjected to SDS- Membrane translocation of the nonreceptor tyrosine kinase PYK-2 PAGE and immunoblotting with isoform-specific Abs (Fig. 7B). has been recently shown to be essential for NK cell cytolysis (32). TIL contain similar levels of membrane-associated PKC compared We compared nonlytic and lytic TIL for levels of PYK-2 protein with control T cells, except PKC-␣ which has higher membrane- (by immunoblotting) and also for PYK-2 subcellular localization by guest on September 24, 2021 associated levels thando control spleen cells. (Because both non- on conjugate formation in vitro (by confocal microscopy). We lytic or recovered TIL have higher PKC-␣ levels, this observation found both lytic and nonlytic TIL have equivalent levels of PYK-2 appears unrelated to lytic status.) On activation in vitro, additional protein, suggesting that down-regulation of PYK-2 expression PKC protein becomes membrane associated. Compared with does not contribute to the TIL lytic defect. Furthermore, and in spleen cells, the kinetics of activation-induced PKC membrane as- contrast to PYK-2 function in NK cells, PYK-2 does not localize sociation in TIL is slightly delayed: by 10 min of stimulation, a to the immunological synapse in fully lytic T cells, thereby ex- portion of PKC in spleen T cells becomes membrane associated; cluding a potential PYK-2 defect in nonlytic TIL (data not shown). whereas a shift in localization does not occur in TIL until after 10 Additionally, the intracellular movement of vesicles to the min of activation in vitro. Because the kinetics of membrane-as- plasma membrane during activation-induced lysis has been re- sociation of PKC is identical for lytic and nonlytic TIL, the delay cently shown to be dependent on the function of a small GTPase, in activation of PKC relative to primary spleen CD8ϩ T cells can- Rab 27a (33, 34). Therefore, we examined nonlytic and lytic TIL not be directly related to defective TIL lysis. This analysis shows for levels (Fig. 9A) and subcellular localization of Rab 27a (Fig. that there is no dramatic difference between nonlytic and lytic TIL 9B). The level of Rab 27a protein is identical in lytic and nonlytic in activation of (several isoforms of) the third protein tyrosine TIL (Fig. 9A), eliminating the possibility that nonlytic TIL do not kinase the function of which is suggested to be required for lytic produce sufficient Rab 27a (or degrade it abnormally), thereby in- activity, PKC. hibiting lytic function. Also, Rab 27a localization was investigated by confocal microscopy (Fig. 9B). Lytic and nonlytic TIL were TIL calcium flux is normal labeled with both anti-Rab 27a (green) and anti-granzyme B (red) Because TCR activation-induced release of lytic granules has been to visualize colocalization of Rab 27a with lytic granules. Rab 27a ϩ shown to be dependent on calcium flux (31), we tested TIL cal- protein is distributed widely in T cells, but granzyme B lytic cium mobilization. Although freshly isolated TIL are nonlytic, cal- granules colocalize with anti-Rab 27a (yellow in Fig. 9B), showing cium flux in response to activation was indistinguishable in com- that nonlytic TIL contain Rab 27a protein which colocalizes with parison with lytic TIL (Fig. 8). Supporting this observation, lytic granules albeit, as shown previously, not at the immunolog- treatment of nonlytic TIL with PMA plus ionomycin did not re- ical synapse. store lytic function (data not shown), showing that although requirement for activation of the proximal signaling pathway was Acquisition of the lytic dysfunction in TIL involves induction of circumvented, TIL lytic function may require another activating a repressor event or may be blocked. Because rapid calcium flux requires Two types of data suggest that acquisition of the lytic dysfunction TCR-dependent signal transduction, this finding further supports in TIL may involve induction of a repressor: signal transduction is the notion that proximal TCR-mediated signaling is intact in TIL. functional in nonlytic TIL (and the levels and localization of other The Journal of Immunology 5049

FIGURE 10. Recovery of lytic function requires proteasome function. TIL were isolated and allowed to recover in vitro for6hinthepresence or absence (DMSO) of the indicated proteasome inhibitors (Freshly-isolated). Downloaded from Some TIL were ﬁrst recovered by plating for 18 h and subsequently incubated with or without inhibitors for 6 h (Recovered). After all incubations, TIL were washed in complete medium and used as effector cells in standard redirected chromium release assays (at an E:T ratio of 10:1 with 51 Na2 CrO4-labeled P-815 cells in triplicate wells) as described in Materials and Methods. Viability of TIL after treatment with inhibitors was equal to that of TIL receiving vehicle only (DMSO). http://www.jimmunol.org/

Discussion Defective lytic function of T cells infiltrating human (9) and rodent (10) tumors has been recognized for years, although the physio- logical basis for this phenotype has not been described. Typically, freshly isolated TIL do not lyse cognate tumor cells or MHC- matched tumor cell lines but can acquire lytic function after puri- by guest on September 24, 2021 fication and propagation in vitro in the presence of FIGURE 9. Lytic and nonlytic TIL contain equivalent amounts of Rab ϩ exogenous IL-2. 27a which colocalizes with granzyme B lytic granules. A, Immunoblot analysis of Rab 27a. Whole cell detergent lysates were prepared from non- We have analyzed the nature of the TIL lytic defect in murine lytic and lytic TIL, separated on SDS-PAGE, and analyzed by immuno- tumors. After purification and brief culture in vitro, TIL demon- blotting with anti-Rab 27a Ab as described in Materials and Methods. B, strate substantial tumor-specific, perforin-mediated cytolysis Rab 27a colocalizes with lytic vesicles in nonlytic and lytic TIL. TIL were showing that TIL are transiently inhibited in lytic function. In isolated and analyzed for localization of granzyme B and Rab 27a on keeping with the activated phenotype demonstrated by expression conjugate formation with cognate tumor cells in vitro by confocal micros- of various cell surface markers, TIL express high levels of FasL copy as described in Materials and Methods. Rab 27a was detected using (12). However, after isolation and recovery in vitro, tumor cell FITC-conjugated secondary Ab, and granzyme B was detected using PE- lysis is exclusively due to exocytosis of lytic granules. FasL-me- conjugated secondary Ab. Arrows, Colocalization of granzyme B and Rab diated killing does not appear to be operative in tumors, because 27a. Magnification, ϫ200. This experiment has been repeated three times. tumor cells are not detectably apoptotic in situ (12). We have investigated a variety of considerations for defective TIL lysis including: TIL are immature (i.e., lacking expression of perforin/serine esterases); packaging of serine esterases into lytic components of the lytic machinery are normal (PYK-2 and Rab granules is defective; or TIL are defective in granule exocytosis. 27a)) and recovery of lytic function does not require protein syn- Importantly, TIL contain high levels of perforin and serine esterase thesis. According to this notion, a putative repressor is degraded proteins that are packaged into lytic granules. These findings argue during TIL recovery in vitro. This was directly tested by perform- against the possibility that, after homing to the tumor site, TIL ing recovery of TIL function in the presence or absence of two require further maturation defined by synthesis and packaging of different inhibitors of proteasome function, lactacystin and MG132 serine esterases into granules. (Fig. 10). If proteasome inhibitors are included during the 6-h in- TIL were considered to possibly resemble memory T cells in cubation in vitro, there is a dose-dependent inhibition of recovery lymphoid organs which are usually nonlytic and require in vitro of lytic function. However, if TIL are cultured in vitro in the ab- stimulation over many days to regain CTL activity (35). However, sence of inhibitor treatment, full lytic activity is recovered, and TIL recover lytic activity after only6hinvitro showing that their addition of proteasome inhibitors after recovery is without effect phenotype is distinct from memory cells. We further considered on lytic function. that TIL may resemble T cells that infiltrate normal nonlymphoid These observations suggest that freshly isolated, nonlytic TIL tissues in terms of their nonlytic phenotype. The best evidence in contain an inhibitor of lytic function the proteasome-dependent support of the notion that peripheral tissue T cells do not behave degradation of which is required to regain lytic activity. like TIL is provided by a recent report from Masopust et al. (36) 5050 DEFECTIVE TIL MTOC MOBILIZATION showing that Ag-specific T cells can exist in an activated form in are demonstrated. Recovery of TIL lytic function and granule lo- tissues and be directly lytic upon isolation. The observation that calization does not require either RNA or protein synthesis, sug- TIL are mature CTL that recover lytic function after removal from gesting that acquisition of the lytic defect does not involve inac- contact with cells within the tumor bed implies that the tumor tivation of a factor(s) required for granule mobilization. Rather, we microenvironment induces the lytic dysfunction. This supposition suggest that a protein(s) is induced in TIL the expression of which is supported by the observation that tumor-draining lymph node prevents MTOC mobilization to the immunological synapse. This CD8ϩ CTL have a high level of tumor-specific lytic capability suggestion is further supported by our observation that inhibition (data not shown) which is obviously lost on entrance to the tumor of proteasome function during recovery in vitro blocks recovery of bed. Despite containing granules loaded with lytic enzymes, mo- lytic function. Because TCR-mediated signal transduction is func- bilization of the lytic granules to the immunological synapse is tional in nonlytic TIL, at least to the point of PI3 kinase-PKC-ERK defective as shown in immunofluorescence microscopic analyses activation (and therein calcium flux), a putative repressor of of TIL:tumor cell conjugates formed in vitro. Defective localiza- MTOC mobilization must act downstream of PI3 kinase-PKC- tion of lytic granules was further shown to be due to the inability ERK activation. Such a repressor could act directly on the MTOC to recruit the MTOC to the immunological synapse, an activation- or may have an unknown target that is required for MTOC acti- dependent event that is required for the proper localization of gran- vation. Supporting this possibility is the observation that in a rat ules and subsequent exocytosis and target cell lysis (24, 37, 38). basophil cell line Ab to CD81 inhibits degranulation without in- Defective MTOC mobilization is not likely due to some inherent hibiting tyrosine phosphorylation or calcium mobilization (42), the defect in expression of tumor Ags by tumor cells in situ, because phenotype demonstrated by nonlytic TIL. That precedent suggests lytic granules do not migrate when conjugates are formed between that a similar regulatory mechanism may exist in TIL, a notion that Downloaded from TIL and Ag-irrelevant P-815 cells by redirected assay. This finding we are at present pursuing. suggests either that some signaling event required for MTOC function is lacking in TIL or that MTOC function is repressed in References nonlytic TIL. 1. Schreiber, H. 1999. Tumor immunology. In Fundamental Immunology. W. Paul, Signaling requirements for CTL degranulation have been stud- ed. Lippincott-Raven, Philadelphia, pp. 1237Ð1270. ied by others where, on the basis of pharmacological inhibitor or 2. Yannelli, J. R., C. Hyatt, S. McConnell, K. Hines, L. Jacknin, L. Parker, http://www.jimmunol.org/ Ag dependency studies, it was shown that TCR-mediated signaling M. Sanders, and S. A. Rosenberg. 1996. Growth of tumor-infiltrating lymphocytes from human solid cancers: summary of a 5-year experience. Int. J. Cancer is required for release of serine esterases (39). On recognition of 65:413. cognate Ag, CTL activate the ras/MAPK/ERK pathway and 3. De Plaen, E., C. Lurquin, B. Lethe, P. van der Bruggen, V. Brichard, therein the PI3 kinase pathway, both of which are apparently re- J. C. Renauld, P. Coulie, A. Van Pel, and T. Boon. 1997. Identification of genes coding for tumor antigens recognized by cytolytic T lymphocytes. Methods 12: quired for cytolytic function (31). Activated PKC has been shown 125. to be required for lytic function in certain T cell clones by influ- 4. Sahin, U., O. Tureci, and M. Pfreundschuh. 1997. Serological identification of encing MTOC mobilization to the immunological synapse (28). human tumor antigens. Curr. Opin. Immunol. 9:709. 5. Pfreundschuh, M. 2000. Exploitation of the B cell repertoire for the identification TIL have defective mobilization of the MTOC despite normal of human tumor antigens. Cancer Chemother. Pharmacol. 46:S3. by guest on September 24, 2021 TCR-mediated calcium flux and PKC activation. Therefore, al- 6. Srivastava, P. K. 2000. Immunotherapy of human cancer: lessons from mice. Nat. though there is a precedent in the literature describing a require- Immunol. 1:363. 7. Finke, J., S. Ferrone, A. Frey, A. Mufson, and A. Ochoa. 1999. Where have all ment for both calcium flux and PKC function in both MTOC func- the T cells gone? Mechanisms of immune evasion by tumors. Immunol. Today tion and T cell cytolysis, the specific defect in TIL must be 20:158. downstream of calcium flux and PKC activation. 8. 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Serological analysis of cell surface antigens of tumors cytolytic granules to the immunological synapse and lytic activity induced by murine leukemia virus. J. Natl. Cancer Inst. 48:265. The Journal of Immunology 5051

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